As is well known, immunoglobulins play an important role in the immune system of mammals. They are produced by B-lymphocytes, found in blood plasma, lymph and other body secretions. Immunoglobulins constitute approximately 20% of the plasma proteins in humans. The basic unit of immunoglobulins is a heterotetramer, containing 2 heavy chains and two light chains, linked by disulphide bonds. Each of these chains have a variable region at their N-terminus which form the antigen binding site, and constant regions, which are responsible for the effector functions of the immunoglobulins.
There are five major classes of immunoglobulins with differing biochemical and physiological properties: IgG (γ heavy chain), IgA (α), IgM (μ), IgD (δ) and IgE (ε). Human IgG represents the most abundant immunoglobulin in plasma, whereas IgA represents the main antibody class in external secretions such as saliva, tears and mucus of the respiratory and intestinal tracts. IgM is by far the physically largest antibody in the human circulatory system, usually being present as a pentamer of the basic immunoglobulin unit, and appears early in the course of an infection.
Initially, IgG preparations from human plasma were successfully used for the prophylaxis and treatment of various infectious diseases. The early products were produced by relatively crude processes (ethanol fractionation), and contained impurities and aggregates to an extent that they could only be administered intramuscularly. Improvements in the purification processes have led to IgG preparations that were suitable for intravenous administration (called IVIG) due to their improved purity and quality, and preparations for subcutaneous administration (called SCIG) have also been developed.
The industrial processes commonly used to purify IgG from plasma are based on the original method devised by Cohn (Cohn E., et al., (1946), J Am Chem Soc, 68, 459-475, Oncley et al., (1949), J Am Chem Soc, 71, 541-550), which dates back to the 1940s and rely on the cold fractionated precipitation of plasma proteins. After progressive additions of ethanol under controlled conditions of ionic strength, pH and temperature, this plasma fractionation process obtains enriched or concentrated fractions of therapeutically useful plasma proteins (coagulation factors, albumin, immunoglobulin, antithrombin III). Applying Cohn's fractionation, IgG is obtained from fractions II+III, I+II+II or the equivalent precipitate A (called NA precipitate) according Kistler and Nitschmann, who developed a modified ethanol fractionation method (Kistler P and Nitschmann H S, (1952), Vox Sang, 7, 414-424).
In the 1960s it was shown that short fatty acids (C6-C12) form insoluble complexes with α- and β-globulins whereas γ-globulins are not as readily precipitated (Chanutin et al., (1960) Arch. Biochem. Biophys. 89; 218).
Steinbuch & Audran ((1969) Arch. Biochem. Biophys. 134, 279-294) described a purification process for IgG with caprylate (i.e. octanoate, a C8-saturated fatty acid) as precipitating agent. Non-immunoglobulins were precipitated from human plasma after dilution with an acetate buffer to reach a final pH of 4.8. After addition of caprylate under vigorous stirring an IgG enriched solution was obtained. The purity and yield depended on the amount of caprylic acid, the pH, the molarity of the buffer and the dilution factor.
Extensive non-immunoglobulin precipitation was best obtained at slightly acidic pH, but not below pH 4.5. Plasma was diluted 2:1 with 0.06 M acetate buffer, pH 4.8, and then treated with 2.5 wt. % caprylate to initiate precipitation. Batch adsorption of the supernatant on DEAE-cellulose was used to clear additional impurities from the isolated IgG fraction. Later work by Steinbuch et al. showed the use of caprylic acid to precipitate most proteins and lipoproteins (other than the immunoglobulins) present in Cohn ethanol Fraction III (Steinbuch et al., (1973), Prep. Biochem. 3, 363-373).
The same method was applied to diluted human plasma using 2.16 wt. % caprylate. (Habeeb et al., (1984) Prep. Biochem. 14, 1-17). Habeeb et al. followed the caprylic acid precipitation with fractionation on DEAE cellulose. The resulting plasma-derived IgG was essentially free of aggregates, plasmin and plasminogen. In addition, the IgG obtained was low in anticomplement activity and relatively stable during storage. The caprylate precipitation step was therefore recognized as very useful, and was introduced into many modern processes for IgG production from plasma.
In addition to the alcohol, PEG and caprylic acid fractionation methods, several chromatographic methods were used in combination with basic fractionation methods for the purification of IVIG.
The most commonly used chromatographic method is ion exchange chromatography which takes advantage of surface distribution and charge density on both the protein and the ion exchange media. The anion exchange resin presents a positively charged surface. The charge density is specific to the resin and generally is independent of pH (within the working range of the resin). A typical anion exchanger will bind proteins which have a net negative charge (i.e. when the pH of the solution is above the isoelectric point of the protein). In reality, the surface of a protein does not present a singular charge; rather it is a mosaic of positive and negative charges, and neutral areas. Surface structure is specific to a given protein and will be affected by solution conditions such as ionic strength and pH. This uniqueness can be exploited to establish specific conditions where individual proteins will bind or release from the anion exchange resin. By establishing these conditions, proteins with only slightly differing surface or charge properties can be effectively separated with high yield (>95%).
Improvements in the structure of chromatography resin supports have made large scale chromatography a practical alternative to more conventional purification methods. Rigid resins allow large volumes to be processed rapidly (<5 hours), and high ligand density gives the increased capacity necessary for large volume processing. These factors coupled with high yields, product purity and process simplicity favor the use of chromatography in large scale manufacturing.
In particular, cation and/or anion exchange chromatography, sometimes combined in separate steps or in series, have been used for purifying IgG from plasma or fractions thereof (e.g. as described in WO 99/64462). In the majority of the methods, anion exchange chromatography is used in negative mode, i.e. conditions are used to enable the binding of the contaminant proteins, e.g. IgA, IgM, albumin, fibrinogen, transferrin, while the IgG is recovered in the non-adsorbed fraction.
The combination of caprylate precipitation followed by ion-exchange chromatography for the purification of IgG was described in many publications. Steinbuch & Audran ((1969) Arch Biochem Biophys 134, 279-284) described the further purification of IgG after precipitation of caprylate with DEAE-cellulose. Lebing et al. (U.S. Pat. No. 5,886,157) described two anion-exchange columns used in series for the removal of IgM, IgA, albumin and other impurities. Lebing et al. combined both caprylate mediated effects, namely the essential reduction of non-IgG proteins by precipitation, thereby using the virus partitioning, and the enveloped virus inactivation properties of the fatty acid in a separate incubation step. The importance of the so-called “pH-swing” starting from the reconstitution of an IgG containing paste/precipitate at pH 4.2 and the subsequent addition of caprylate upon adjusting the pH 5.2 is stressed to be essential for the IgG enriching procedure, thus needed to effectively reduce non-IgG proteins. A few other impurities, like IgA and IgM, as well as the caprylate were subsequently reduced by the mentioned ion exchange chromatography steps.
U.S. Pat. No. 5,164,487 concerns the use of caprylic acid for the manufacture of an intravenously tolerable IgG preparation free from aggregates, vasoactive substances and proteolytic enzymes. The method includes contacting the starting material containing IgG with 0.4% to 1.5% caprylic acid before chromatographic purification with an ion exchange or hydrophobic matrix.
Due to the continuous improvements in purification processes, there has been an evolution in IgG products over the years. As mentioned above, the first IgG products were only suitable for intramuscular use, as they caused too many adverse events when administered intravenously. The first generation of an IgG product suitable for intravenous use (IVIG) was prepared by pepsin cleavage of the starting material (Cohn fraction II), the purpose of the cleavage being removal of immunoglobulin aggregates which caused serious adverse events such as complement activation and made it impossible to administer the early products intravenously. No column chromatography steps were included in the process. The product had to be freeze-dried in order to remain stable for a reasonable period of time and was dissolved immediately prior to use.
A second generation of IVIG based on uncleaved and unmodified immunoglobulin molecules with low anticomplement activity and higher stability was introduced in the mid-eighties, but still in the form of a freeze-dried product. This IVIG was purified by processes including several chromatography steps. Pepsin cleavage was avoided, aggregates and particles were removed by precipitation, and further purification was achieved by column chromatographic ion exchange methods.
For the third generation of IVIG, dedicated virus inactivation steps were included in the processes. While particularly the precipitation steps in the purification processes removed a lot of viruses, some patients treated with blood products nevertheless were infected with HIV, necessitating further, dedicated steps to be taken to inactivate and remove viruses from these products.
The processes were continued to be refined further to achieve better purity and quality of the protein, in order to enable stable liquid products to be made available, and to improve the safety and tolerability of these products for patients. In addition, subcutaneous formulations were developed.
IgG products are now used in a number of clinical applications. In addition to the traditional use for the treatment of primary or acquired immunodeficiencies, and infectious diseases, it has been shown that these products are also effective in the treatment of autoimmune diseases and certain neurological disorders such as CIDP. There has also been a marked increase in the number of studies focusing on further therapeutic uses of IgG products. Thus the demand for IgG products has been increasing. IgG products are now the plasma products in greatest demand on the world market; in 2008 the market reached approximately 82 metric tons (including 37 tons in the USA, 21 tons in Europe and 17 tons in Asia) with a tendency to grow at a rate of approximately 7% a year (the predicted demand in 2013 is 110 metric tons) (The Worldwide Plasma Fractions Market 2008. The Marketing Research Bureau, Inc. April 2010 Edition). As human plasma is a valuable, limited resource, the processes for purification of IgG from plasma need to be further improved to achieve higher yields than currently possible while not compromising the quality of the product. Current processes have an average yield of 3.7 to 4.2 g of IgG per liter of plasma, which represents only up to 55% of the IgG present in plasma.